Role Of Lyn Kinase In The Pathogenesis Of Chronic Lymphocytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 668-668
Author(s):  
Phuong-Hien Nguyen ◽  
Nina Reinart ◽  
Michael Hallek
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Lymphocytic Leukemia ◽  
Negative Regulator ◽  
Lymphoid Tissues ◽  
Malignant Cells ◽  
Deficient Mice

Abstract The Src family kinase Lyn is predominantly expressed in B cells and plays a central role in initiating B cell receptor (BCR) signaling. Lyn is associated with BCR complexes and is renowned for its role in B cell activation and proliferation. Active Lyn contributes to positive regulation of signalling through tyrosine phosphorylation of components of the BCR. Intriguingly, Lyn was also shown as a negative regulator of BCR signal transduction. Lyn plays an essential role in negative regulation of signalling through its unique ability to phosphorylate immunoreceptor tyrosine based inhibition motifs (ITIM) in inhibitory cell surface receptors. ITIM phosphorylation induces the recruitment of inhibitory phosphatases such as SHP-1/2 and SHIP-1, which attenuate BCR signalling. Lyn-deficient mice have reduced number of B cells and increased numbers of myeloid progenitors. It was reported that expression and activity of Lyn in human chronic lymphocytic leukemia (CLL) is elevated compared to healthy B cells. Besides, higher levels of Lyn are associated with a shorter treatment-free survival of CLL patients. This rises up a hypothesis about Lyn’s significant role in B cell tumorigenesis, malignant transformation of B cells, and the balance between myeloid cells and B lymphocytes. We generated Eµ-TCL1 transgenic LYN-deficient mice (TCL1+/wtLYN-/-) and monitored them in order to identify the population of malignant B cells and to characterize the development of malignant cells in these mice in comparison with Eµ-TCL1 transgenic mice (TCL1+/wtLYNwt/wt). In comparison to TCL1+/wtLYNwt/wt mice, TCL1+/wtLYN-/- mice show a significantly reduced number of malignant B cells in the peripheral blood, as well as a reduced leukocyte count. Besides, TCL1+/wtLYN-/- mice have significantly decreased infiltration of malignant B cells in lymphoid tissues such as spleen, liver, lymph node and bone marrow. This result is also resembled in a hepato-splenomegaly in the TCL1+/wtLYNwt/wt mice. These mice develop severe splenomegaly and hepatomegaly due to infiltration of malignant cells, while TCL1+/wtLYN-/- mice do not develop hepatomegaly. The non-transgenic LYN-/- control mice develop splenomegaly due to infiltration of myeloid cells. Although TCL1+/wtLYN-/- mice have hindered development of TCL1-induced CLL, preliminary data suggest it is not only due to LYN-deficiency in B cell compartment of these mice. Indeed, B cell of TCL1+/wtLYN-/- mice show enhanced proliferation and better survival ex vivo compared to TCL1+/wtLYNwt/wt mice. Notably, TCL1+/wtLYN-/- mice developed a skewed microenvironment which might contribute to CLL down regulation. LYN-/- microenvironment, particularly in aged mice, does not support engraftment of TCL1-induced leukemic B cell as well as LYNwt/wt mice in our transplantation model. These results point to a complex regulation of Lyn signalling in CLL involving not only leukemic cells but also cells of the micromillieu, that needs further investigation. Disclosures: No relevant conflicts of interest to declare.

Overexpression of the CXCR5 chemokine receptor, and its ligand, CXCL13 in B-cell chronic lymphocytic leukemia

Blood ◽  
2007 ◽  
Vol 110 (9) ◽  
pp. 3316-3325 ◽  
Author(s):  
Andrea Bürkle ◽  
Matthias Niedermeier ◽  
Annette Schmitt-Gräff ◽  
William G. Wierda ◽  
Michael J. Keating ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Actin Polymerization ◽  
Serum Levels ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Accessory Cells ◽  
Mitogen Activated Protein ◽  
B1 Cells

Abstract CXCL13 is a homeostatic chemokine for lymphocyte homing and positioning within follicles of secondary lymphoid tissues, acting through its cognate receptor, CXCR5. Moreover, the CXCR5-CXCL13 axis plays a unique role in trafficking and homing of B1 cells. Here, we report that chronic lymphocytic leukemia (CLL) B cells express high levels of functional CXCR5. CXCR5 expression levels were similar on CLL B cells and normal CD5+ B cells, and higher compared with normal CD5− B cells, follicular B-helper T cells (TFH cells), or neoplastic B cells from other B-cell neoplasias. Stimulation of CLL cells with CXCL13 induces actin polymerization, CXCR5 endocytosis, chemotaxis, and prolonged activation of p44/42 mitogen-activated protein kinases. Anti-CXCR5 antibodies, pertussis toxin, and wortmannin inhibited chemotaxis to CXCL13, demonstrating the importance of Gi proteins and PI3 kinases for CXCR5 signaling. Moreover, CLL patients had significantly higher CXCL13 serum levels than volunteers, and CXCL13 levels correlated with β2 microglobulin. We detected CXCL13 mRNA expression by nurselike cells, and high levels of CXCL13 protein in supernatants of CLL nurselike cell cultures. By immunohistochemistry, we detected CXCL13+ expression by CD68+ macrophages in situ within CLL lymph nodes. These data suggest that CXCR5 plays a role in CLL cell positioning and cognate interactions between CLL and CXCL13-secreting CD68+ accessory cells in lymphoid tissues.

CCR4:CCL17 Interaction Influences TLR-9 Mediated Cell Survival and Proliferation In Chronic Lymphocytic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3593-3593
Author(s):  
Sonal C. Temburni ◽  
Ryon M. Andersen ◽  
Luke Janson ◽  
Xiao-Jie Yan ◽  
Barbara Sherry ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Dose Range ◽  
Serum Levels ◽  
Lymphocytic Leukemia ◽  
Novel Therapy

Abstract Abstract 3593 Unlike other hematologic disorders, chronic lymphocytic leukemia(CLL) exhibits remarkable heterogeneity in the rates of disease progression among cases. CLL cells survive by receiving signals from the microenvironment via various receptors: B-cell antigen receptor (BCR), Toll-like receptors (TLRs) and cytokine and chemokine receptors. We previously reported that CLL clones with somatically mutated IGHVs and high (≥30%) percentage of CD38 expressing cells have the highest percentage of CCR4-expressing cells. To further explore the functional contribution of the CCR4:CCL17 axis in CLL, we studied CCL17-induced chemotactic behavior in 16 CLL cases. In transwell cultures we observed a bimodal migratory response to CCL17 at 2 doses in a dose range of 0.78– 25ng/ml, in ~60% of cases; the remaining cases showed maximal migration at a single dose (1.56 or 3.12ng/ml). A comparison of phenotypes of the migrated and non-migrated cell populations was undertaken in 10 cases, analyzing CXCR3, CXCR4, CCR4 and CCR7 that are involved in homing of cells to sites favoring growth, and CD31, CD38 and CD69, activation related molecules. The migrated cells consistently showed significantly higher percentages and densities of CD38 expression than the non-migrated cells suggesting a role for CD38 in the CCR4-mediated downstream pathway. CCR4 ligand, CCL17, is constitutively expressed in the thymus and is produced by dendritic cells, endothelial cells, keratinocytes and fibroblasts, whereas CCL22 is produced by tumor cells and the tumor microenvironment. Serum levels of both these ligands in untreated patients were quantified by ELISA. CCL17 levels ranged between 45-1, 229 pg/ml in U-CLL cases (n=23) and between 43-1, 418 pg/ml in M-CLL cases (n=30). CCL22 levels ranged between 121-5, 497 pg/ml in U-CLL cases (n=23) and 409-5, 502 pg/ml in M-CLL cases (n=30). The percentages of CCR4- expressing B cells directly correlated with percentages of T cells expressing CCR4 in individual cases, whereas they inversely correlated with both, serum levels of CCL17 (p< 0.01) and CCL22 (p< 0.05). CCL17 produced by DCs in peripheral organs may exert an accessory role in BCR- and TLR-9-mediated immune responses in B cells. We therefore tested if CCL17 supported BCR- and TLR-mediated proliferative responses in a cohort of 31 (16 U-CLL and 15M-CLL) CLL cases. CCL17 augmented BCR-mediated B-cell proliferation in 9/16 (56%) U-CLL cases, but only in 3/15 (20%) M-CLL cases. On the other hand, CCL17 showed an additive effect in promoting TLR-9-mediated cell proliferation in 13/15 (87%) M-CLL cases at a dose of 2ng/nl (approximating that detected in serum); it also augmented TLR-9 mediated B cell proliferation in 6/16 U-CLL cases but at a 5-fold or higher dose (10-25 ng/ml). In a subset of this cohort (8 cases) CCL17-induced modulation of molecules involved in the apoptotic process was studied. We found upregulation of anti-apoptotic proteins Mcl-1 and Bcl2 and down-regulation of pro-apoptotic molecules Bim, PUMA, and Bid in 5 of these cases. The pro-survival effects of CCL17 were partially abrogated by the blocking anti-CCR4 mAb (1G1). Taken together, these findings suggest that CCL17 plays a role in modulating TLR-9-mediated signaling and migration in CLL. Therefore, inhibition of CCR4:CCL17 interaction in vivo represents a novel therapy by preventing migration of CLL cells towards an environment that promotes their survival. Disclosures: No relevant conflicts of interest to declare.

B Cell Stimulation through BCR and CD40 Modulates the Response of Chronic Lymphocytic Leukemia Cells to BAFF and APRIL.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1361-1361
Author(s):  
Gerardo Ferrer ◽  
Kate E Hodgson ◽  
Victor Ciria ◽  
Gael Roue ◽  
Dolors Colomer ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Activation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Lymphocytic Leukemia ◽  
Receptor Expression ◽  
Cd40 Ligation ◽  
Stimulation Of

Abstract Abstract 1361 The two TNF family proteins (B-cell activating factor [BAFF] and a proliferation-inducing ligand [APRIL]) and their three receptors (transmembrane activator and CAML interactor [TACI], B-cell maturation antigen [BCMA], and BAFF receptor [BAFF-R]) play a critical role in the process of differentiation, maturation and survival of normal B cells. Additionally, recent studies indicate that activation or inhibitory signals can modulate the sensitivity of normal B cells to BAFF and APRIL through the regulation of their receptors. In chronic lymphocytic leukemia (CLL), BAFF and APRIL have been shown to increase survival of neoplastic B cells in vitro. We investigated whether stimulation of CLL cells through the B cell receptor (BCR) or CD40 ligation could regulate the expression of BAFF-R, TACI and BCMA and enhance BAFF and APRIL sensitivity. Purified B cells were obtained from 23 CLL patients and nine healthy controls. Receptor expression was measured by flow cytometry at baseline and at 48 hours after stimulation with F(ab’)2 antihuman IgM (10 μg/ml) and CD40L (500ng/ml) plus IL-4 (20ng/ml). Cell activation and viability, as assessed by labeling CD69 and Annexin V/TO-PRO-3, were evaluated at 48, and at 72 hours after co-stimulation with either soluble BAFF (100ng/ml) or APRIL (500ng/ml). Baseline analyses showed that BAFF-R was the most highly expressed receptor in CLL cells and normal B cells (Mean fluorescence intensity (MFI) ratios, 213.5 and 185.8, respectively). TACI and BCMA were also expressed in all CLL cells and normal B cells (MFI ratios TACI: 2.5 and 1.9; BCMA: 14.8 and 6.6, respectively), but at a significantly lower level than BAFF-R (p<0.001). Furthermore, BCMA MFI ratio was significantly higher in CLL than in normal B cells (p=0.015). After 48h of culture, an increase of all three receptors was observed in normal B cells in response to either BCR stimulation or CD40 ligation. In contrast, in CLL cells BCR stimulation induced almost no variation in the receptors expression in all cases. This was accompanied by a failure of cell activation and a significant decreased viability of CLL cells (from 36% to 24% p=0.013). By contrast, CD40 ligation in CLL cells induced a significant upregulation of TACI expression (p=0.007) and a significant reduction of BCMA (p=0.007), which correlated with an increase of CLL cell activation and viability (p<0.001). BAFF-R levels did not change. The addition of exogenous soluble BAFF or APRIL showed increase in the viability of normal B cells at 72 hours independently of whether cells were unstimulated or stimulated through the BCR or by CD40 ligation. In CLL cells, however, the viability was significantly increased in CD40-stimulated cells whereas in either unstimulated or BCR-stimulated CLL cells, the addition of BAFF and APRIL had a modest effect on viability (Table). These findings indicate that stimulation of CLL cells through the BCR and CD40 modifies the sensitivity of CLL cells to respond to BAFF and APRIL which reflects the regulation of BCMA, TACI and BAFF-R. In contrast to normal B cells, CD40-ligation in CLL cells upregulated only TACI expression. The fact that the addition of CD40L plus IL-4 and BAFF increased viability in CLL cells while BAFF alone had almost no effect may be related to the ability of CD40 ligation to increase TACI expression. Although BCR stimulation failed to increase the expression of the receptors, co-stimulation by BAFF plus BCR increased viability in CLL cells. Disclosures: No relevant conflicts of interest to declare.

IL4 and CD40L Prevent Apoptosis of Chronic Lymphocytic Leukemia Cells Via Intracellular pSTAT6 and NFkB Signaling and Not Via Receptor Kinetics

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3893-3893
Author(s):  
Daniel Mertens ◽  
Nupur Bhattacharya ◽  
Sarah Häbe ◽  
Hartmut Döhner ◽  
Stephan Stilgenbauer
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Conditioned Medium ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Malignant Cells ◽  
Downstream Signaling ◽  
Receptor Kinetics

Abstract Abstract 3893 Chronic lymphocytic leukemia (CLL) cells are highly dependent on microenvironmental input for their extended survival in vivo, but the underlying molecular mechanism is still unclear. Compared to non-malignant B-cells, CLL cells are more responsive to contact dependent complex stimuli like coculture on bone marrow derived stromal cell lines of both human (p<0.0001) and murine origin (p<0.01), but also to soluble factors (human conditioned medium p<0.0001, murine conditioned medium p<0.001, all student′s t-test). In order to understand the intrinsic difference of the anti-apoptotic phenotype of CLL cells, the signalling circuitry of the malignant cells was modelled. Compared to candidate ligands like SDF-1 (at concentrations between 10–1000ng/ml), BAFF (250–1000ng/ml), APRIL (250–1000ng/ml) and soluble anti-IgM (1–25μg/ml), the factors CD40L (10–2000ng/ml) and IL4 (0.1–10ng/ml) were the most efficient ligands in rescuing CLL cells from spontaneous death in vitro. The dose response of IL4 and CD40L displayed different saturation and cooperativity between CLL cells and non-malignant B-cells. Using IL4, saturation was reached both for CLL cells and B-cells at 0.2pM, but at 52% survival (+/− 8%) for CLL cells and 28% (+/−7%) for B-cells, and the estimated dissociation constant Kd was 0.01pM for both ligands. For CD40L, CLL cell survival reached saturation at 40nM, while no saturation was reached for B-cells. Intriguingly, B-cells showed cooperativity in their response to CD40L, with a cooperativity coefficient of 2.0 and a Kd of 70pM, while cooperativity for CD40L was lost in CLL cells (Kd of only 2.6pM). This pointed towards distinct differences in ligand-receptor interactions or in downstream signaling between CLL cells and non-malignant B-cells. However, high-throughput spatial analysis with a microscope-coupled cytometer did not show differences of receptor quantity or receptor distribution between malignant and non-malignant cells. In contrast, quantity and phosphorylation levels of downstream signalling nodes like STAT6 (measured by flow cytometry and validated by Western-blot) and the activity of NF-kB (p65 binding to DNA measured by oligonucleotide-coupled ELISA) were higher in CLL cells compared to B-cells from healthy donors. Therefore, the defect in IL4 and CD40L signalling that leads to an enhanced survival in CLL cells is likely caused by changes in the intracellular circuitry. Disclosures: No relevant conflicts of interest to declare.

scholarly journals NFkB p50 (Nfkb1) Contributes to Disease in the Eu-TCL1 Mouse Model of Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1248-1248
Author(s):  
Erin K Hertlein ◽  
Timothy L Chen ◽  
David M Lucas ◽  
Nikhil Gupta ◽  
Jessica MacMurray ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Mouse Model ◽  
Gene Silencing ◽  
Disease Progression ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Negative Regulator ◽  
Intermediate Phenotype ◽  
Lymphoid Tissues ◽  
High Degree

Abstract Chronic lymphocytic leukemia (CLL) is a disease of mature B-cells that are resistant to apoptosis and accumulate in the blood over time. Due to the slowly progressing nature of this disease, studies to determine the molecular events leading to defective apoptosis are challenging. Our group has previously reported that during disease progression in the Eμ-TCL1 CLL mouse model, genes become silenced in a progressive manner over the course of the disease. This silencing is due in part to a high degree of methylation at the promoters of key tumor suppressors. However, even though a high degree of methylation is observed in both this mouse model and in CLL patient samples, agents targeting methylation such as decitabine have proven ineffective in CLL therapy. Therefore other novel methods to reverse gene silencing in CLL are an attractive therapeutic option. In our previous study, we observed early transcriptional mechanisms of gene silencing (occurring prior to methylation) involving NF-κB p50 homodimers. NF-κB is a family of transcription factors which is known to play an important role in the progression of CLL. Advances in next generation sequencing have recently identified loss of function mutations in NFKBIE,a negative regulator of NF-κB signaling,in CLL. These mutations contribute to increased nuclear localization (and hence increased activation) of NF-κB subunits. In addition, a mutagenesis screen in the Eμ-TCL1 mouse found that several of the most frequently occurring mutations that contribute to disease progression occur in genes related to the NF-κB family, particularly in the p50 (Nfkb1) gene. In the present study, we have generated a new mouse model to further study the role of p50 in CLL pathogenesis. The Eμ-TCL1 mouse was crossed to the p50-/- mouse. The p50-/- mice are fertile with normal growth and development under sterile conditions; however they do exhibit a defective response to infection and decreased antibody production. As such, animals in this study are housed under pathogen free conditions. The initial cross resulted in mice that were all heterozygous for p50 (p50+/-), and either positive or negative for the TCL1 transgene. The p50+/-; TCL1+ animals were subsequently crossed with one another to generate three genotypes: p50+/+. p50+/- and p50-/-. Animals are monitored for disease by monthly flow cytometry analysis of CD19 and CD5 in whole blood, and leukemia is defined as >10% double positive cells. The p50-/- mice have a significantly lower incidence of leukemia compared to p50+/+ mice (Chi-square p <0.001), and p50+/- mice show an intermediate phenotype (p=0.024 compared to p50-/- mice). Despite pathogen free conditions, some mice within the p50-/- group die at an early age with no evidence of disease. Therefore, competing risks regression was performed to take into account the mice that die without leukemia via the CIF (cumulative incidence function) based on the Fine and Gray model. Differences in time to leukemia between the p50+/+ and p50-/- mice remained significant (Subdistribution Hazard Ratio; SHR = 8.45; 95% CI: 1.86 - 38.47; p=0.006). The p50+/- mice still exhibit an intermediate phenotype, with more separation between the p50+/- and p50-/- (SHR = 4.39; 95% CI: 1.00 - 19.29; p=0.050) than the p50+/- and p50+/+ groups (SHR = 0.52; 95% CI: 0.26 - 1.06; p=0.069). Finally, spleens from p50-/- mice are notably smaller than the p50+/+ littermates (average spleen score of 0-1 for p50-/- versus 2-3 for p50+/+, using a 0-4 scale), indicating that disease in the secondary lymphoid tissues is also affected by the loss of p50 in this model. In conclusion, these data genetically demonstrate the significant contribution of the p50 (Nfkb1) gene to disease progression in the Eµ-TCL1 mouse model. These studies highlight the importance of the NF-κB family in CLL, and suggest that p50 is a promising therapeutic target in this disease. Disclosures No relevant conflicts of interest to declare.

Inhibition of JAK2/STAT3 Pathway Leads to Apoptosis in Chronic Lymphocytic Leukemia Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2023-2023 ◽  
Author(s):  
Filippo Severin ◽  
Federica Frezzato ◽  
Veronica Martini ◽  
Flavia Raggi ◽  
Valentina Trimarco ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Viability ◽  
B Cell ◽  
B Lymphocytes ◽  
Sh2 Domain ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Stat3 Phosphorylation ◽  
Jak2 Inhibition

Abstract INTRODUCTION Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature clonal CD19+/CD5+/CD23+ B lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. Despite their in vivo prolonged lifespan due to intrinsic defects, CLL leukemic cells rapidly undergo spontaneous apoptosis in vitro, highlighting the need of extrinsic signals delivered by the microenvironment. Several molecules, including those released by mesenchymal stromal cells (MSCs), signal through JAK (Janus kinases)-STAT (Signal Transducers and Activators of Transcription) pathways. We particularly focused on the JAK2/STAT3 axis since Interleukin-6 (IL-6), one of the most abundant cytokines released in the CLL microenvironment, is the key ligand of the receptor triggering this pathway. The deregulation of JAK2/STAT3 axis may lead to aberrant activation of STAT3 and, as a result, to tumor development in hematopoietic cells. METHODS B cells were collected from 12 controls and 46 CLL patients. Purified cells (2x106cells/ml) were cultured, and treated with AG490 (10, 50 and 100μM), AZD1480 (1, 4 and 10μM), Fedratinib (1, 5 and 10μM), and Ruxolitinib (0.313, 2.5 and 10μM) (which are JAK2 inhibitors), and the STAT3 inhibitor Stattic (5, 7.5, and 10μM) for 24, 48 and 72h. Experiments with AG490 and Stattic were performed with/without MSCs. STAT3 expression and phosphorylation were evaluated by Western Blotting (WB) and Flow Cytometry (FC), and its localization was analyzed by confocal microscopy and subcellular fractionation. CLL and normal B cell viability was tested by FC with Annexin V/PI test. RESULTS We demonstrated that STAT3 was highly expressed in malignant B cells with respect to normal B lymphocytes. As far as STAT3 phosphorylation at Tyr705, that is an essential step for STAT3 activation, we demonstrated a constitutive phosphorylation in CLL cells by FC and WB analyses, although in some patients STAT3 Tyr705 phosphorylation is barely detected. We also pointed out that the in vitroincubation of leukemic B cells with AG490 and Stattic and Fedratinib, induces a dose-dependent apoptosis of CLL B cells. However, the tested doses of Ruxolitinib and AZD1480 did not seem to CLL B cell viability but only STAT3 phosphorylation. Both AG490 and Stattic were able to bypass the microenvironmental protection when neoplastic B cells were co-cultured with MSCs. STAT3 Tyr705 localization was analyzed in normal and leukemic B cells by a subcellular protein fractionation. We separated nuclei from cytosol, detecting STAT3 Tyr705 both in the cytosolic and in the nuclear fractions of CLL B cells. We showed that AG490 and Fedratinib treatment on CLL cells can mediate other effects: i) SHP-1 activity is turned on by JAK2 inhibition, decreasing its phosphorylation at Ser591; ii) AG490 administration inactivates protein Lyn, reducing the phosphorylation in its active site at Tyr396. Lyn, a Tyr-kinase, and SH2-domain containing Tyrosine Phosphatase (SHP-1), a Tyr-phosphatase, are both involved in the prolonged lifespan of neoplastic CLL cells. To confirm the link between JAK2 inhibition by AG490 and Lyn dephosphorylation, we added sodium orthovanadate (Na3VO4, 100 μM), a phosphatase inhibitor, to cell culture to restore Lyn activation (resulting from the inactivation of SHP-1 phosphatase); as a result, Lyn Tyr396 phosphorylation was restored. On the contrary, the treatment of CLL cells with Stattic did not induce any change in SHP-1 status with respect to untreated cells since Stattic is effective on STAT3, that is a downstream protein with respect to JAK2. Since Stattic did not affect SHP-1 activation, it does not impact on Lyn activation/phosphorylation. CONCLUSIONS The ability of AG490 and Stattic to induce apoptosis in leukemic B cells bypassing the pro-survival stimuli provided by the tumor microenvironment and the Fedratinib effectiveness at low doses, represents a starting point for the development of new therapeutic strategies in CLL. This study also provides new insights for the investigation of the pathogenesis of CLL focusing the attention on the cross-talk between JAK/STAT and BCR/Lyn axes. Disclosures No relevant conflicts of interest to declare.

Impaired Expression of p66Shc, a Novel Regulator of B-Cell Survival, in Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 801-801
Author(s):  
Cosima T. Baldari ◽  
Nagaja Capitani ◽  
Orso Maria Lucherini ◽  
Elisa Sozzi ◽  
Micol Ferro ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Apoptosis ◽  
Conflicts Of Interest ◽  
Group Analysis ◽  
Lymphocytic Leukemia ◽  
Expression Of Genes ◽  
Mutational Status ◽  
Healthy Donors

Abstract Abstract 801 Intrinsic defects in the apoptotic circuitry underlie to a large extent the extended survival of malignant B cells in chronic lymphocytic leukemia (CLL) and are moreover believed to be responsible for their resistance to chemotherapy. We have recently demonstrated that p66Shc, a member of Shc family of protein adapters, acts as a promoter of apoptosis in T cells. Here we show that p66Shc uncouples the B-cell antigen receptor (BCR) from the Erk and Akt dependent survival pathways, thereby enhancing B-cell apoptosis. Expression of p66Shc was found to be profoundly and consistently impaired in CLL B cells compared to peripheral blood B cells form healthy donors. Moreover, significant differences in p66Shc expression were observed in patients with favorable or unfavorable prognosis, classified on the basis of the mutational status of the IGHV genes, with the lowest expression in the unfavorable prognosis group. Analysis of the expression of genes previously implicated in the apoptosis defects of CLL B cells revealed a selective alteration in the balance of pro- and anti-apoptotic members of the Bcl-2 family in these patients. Reconstitution experiments in CLL B cells, as well as data obtained on B cells from p66Shc-/- mice, showed that p66Shc expression correlates with a bias in the Bcl-2 family towards the pro-apoptotic members. Collectively, the data identify p66Shc as a novel regulator of B cell apoptosis which attenuates survival signals emanating from the BCR and modulates expression of the Bcl-2 family. They moreover provide evidence that the defect in p66Shc expression identified in CLL B cells may be causally related to the imbalance towards the anti-apoptotic Bcl-2 family members observed in these cells. Disclosures: No relevant conflicts of interest to declare.

Preclinical Development Of ROR1 Peptide Based Vaccine With Activity Against Chronic Lymphocytic Leukemia In ROR1 Transgenic Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4174-4174 ◽  
Author(s):  
Jian Yu ◽  
Bing Cui ◽  
George F. Widhopf ◽  
Liguang Chen ◽  
Laura Rassenti ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
High Titer ◽  
Lymphocytic Leukemia ◽  
Orphan Receptor ◽  
Lymphoid Tissues ◽  
Mouse Tissues ◽  
Freund’S Adjuvant ◽  
Freund's Adjuvant

Abstract Receptor tyrosine kinase-like orphan receptor (ROR1) is expressed on chronic lymphocytic leukemia (CLL) and other cancers, but not on normal post-partum tissues, except for a small subset of precursor B cells known as hematogones. Because of its restricted expression on cancer cells, ROR1 is an attractive target for developing novel anti-cancer therapies. In this study, we designed several different peptides, corresponding to distinct epitopes in the extracellular domain of ROR1. Each peptides was conjugated with keyhole-limpet hemocyanin (KLH) to generate a peptide-KLH vaccine, which we used to immunize C57BL/6 mice, first in complete Freund’s adjuvant (CFA) and then again 2 weeks later in incomplete Freund’s adjuvant (IFA). Two weeks following the second injection the sera from immunized mice were tested for binding activity for recombinant ROR1 protein via immunoblot analyses or ELISA or for cell-surface ROR1 by flow cytometry. We identified one peptide-KLH vaccine (R22-KLH) that induced high-titer anti-ROR1 antisera specific for ROR1-expressing cells that did not react with normal human or mouse tissues, which did not express this protein. The antisera generated by mice immunized with R22-KLH were capable of directing specific complement-dependent cytotoxicity (CDC) against neoplastic cells that expressed ROR1 (e.g. human CLL cells, EW36, or JeKo-1), but not against cells that did not express ROR1 (e.g. Jurkat). To study the capacity of R22-KLH to break self-tolerance, we immunized C57BL/6 mice transgenic for human ROR1 controlled by the immunoglobulin μ heavy-chain promoter/enhancer, designated as ROR1-Tg mice. Despite having B cells that express surface ROR1, such animals still were able to generate high-titer anti-ROR1 antisera comparable in binding and CDC activity and specificity as antisera generated in C57BL/6 mice. Production of such antisera was associated with reduced expression of ROR1 by the B cells of such ROR1-Tg mice. Cohorts of C57BL/6 mice or ROR1-Tg mice were immunized with R22-KLH or KLH and then challenged with intravenous injections of 3 × 105 or 2 × 104 ROR1-expressing primary CLL cells of double-transgenic ROR1xTCL1 C57BL/6 mice. Immunization with R22-KLH, but not KLH, significantly inhibited leukemia engraftment, as demonstrated by evaluation of the spleen and lymphoid tissues 4 weeks after adoptive transfer. To generate vaccines that might be suitable for clinical studies, we examined the relative immunogenicity of R22-KLH mixed with newly-developed agonists of toll-like receptor 4 (TLR-4; 1Z105) and TLR-7 (1V270). For this, we compared the immune responses of C57BL/6 mice or ROR1-Tg mice to R22-KLH administered s.c. in CFA/IFA to that of R22-KLH administered s.c. with 1Z105/1V270 in saline. Either C57BL/6 or ROR1-Tg mice generated higher titers of anti-ROR1 antibodies following immunization with R22-KLH in 1Z105/1V270 than did animals immunized with R22-KLH in CFA/IFA. Moreover, injection of R22-KLH in 1Z105/1V270 appeared better tolerated than injection of R22-KLH in CFA/IFA. These data indicate that immunization with R22-KLH can break tolerance to ROR1 and induce high-titer anti-ROR1 antisera. The immunogenicity of R22-KLH can be enhanced by injection with agonists of TLR4/7, obviating use of CFA/IFA. Pharmacology/toxicology studies are ongoing to determine the suitability of R22-KLH in 1Z105/1V270 for clinical trials involving patients with ROR1-expressing cancers. Disclosures: No relevant conflicts of interest to declare.

Overexpression of the CXCR5 Chemokine Receptor, and Its Ligand, B Cell-Activating Chemokine-1 (BCA-1/CXCL13) in B Cell Chronic Lymphocytic Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2948-2948
Author(s):  
Andrea Bürkle ◽  
Jan A. Burger
Keyword(s):  
Dendritic Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Healthy Volunteers ◽  
B Lymphocytes ◽  
Surface Expression ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Follicular Dendritic Cells

Abstract The chemokine B cell-activating chemokine-1 (BCA-1/CXCL13) is an important homing factor for lymphocytes to B cell zones of secondary lymphoid tissues. CXCL13 acts through its cognate receptor, CXCR5. Normal, mature B cells and a subset of memory T cells express CXCR5 chemokine receptors and migrate in response to BCA-1. However, BCA-1 displays a preferential chemotactic activity for B1 B cells when compared to “normal” B2 B cells. Because B lymphocytes from patients with Chronic Lymphocytic Leukemia (B-CLL) are in several aspects comparable to murine B1 cells, we hypothesized that the CXCR5-CXCL13 axis may be highly active in CLL. Initially, we noticed that CLL cells express functional CXCR5 receptors that induce actin polymerization, CXCR5 endocytosis, chemotaxis, and a prolonged activation of p44/42 MAP kinases. In addition, we examined CXCR5 surface expression in a series of CLL patients by flow cytometry and compared the results with normal B cells, or other leukemic B cell lymphoma. In CLL, leukemia B cells expressed significantly higher surface expression of CXCR5 (mean fluorescence intensity ratio/MFIR: 121 ± 9 (±SEM), n = 26) than circulating, CD19 positive B cells from healthy volunteers (CXCR5-MFIR: 69.9 ± 5.4, n = 11, p = 0.002). Neoplastic B cells from other leukemic B cell lymphomas displayed low surface CXCR5 expression (MFIR 19.7 ± 5.9, n = 11). Serum levels of CXCL13 were evaluated by ELISA. Sera from CLL patients displayed significantly higher levels of CXCL13 (mean ± SEM: 170.1 ± 21.5 pg/ml, n = 22) when compared to sera from healthy volunteers (mean ± SEM: 70.7 ± 5.2 pg/ml, n = 10, p = 0.004). Follicular dendritic cells (FDC) have been considered the main source of CXCL13 in secondary lymphoid tissues, thereby attracting T and B lymphocytes for cognate interactions. Surprisingly, we did not detect significant levels of CXCL13 in supernatants of HK follicular dendritic cells, that previously were demonstrated to protect CLL cells from apoptosis (Pedersen &Reed, Blood.2002;100:1795–801). In contrast, high levels of CXCL13 were detected in supernatants of CLL cell cultures in the presence of nurselike cells (NLC). In NLC cultures, CXCL13 levels were 610 ± 129.8 pg/ml (mean ± SEM, n = 4), whereas FDC supernatants contained 0.22 ± 0 pg/ml CXCL13 (mean ± SEM, n = 2). Because of these high CXCL13 levels in NLC cultures, we examined CXCR5 downregulation on CLL B cells in NLC co-cultures. When compared to freshly isolated CLL B cells, CLL cells from NLC cultures express significantly lower surface CXCR5. CXCR5 MFIR of CLL cells from NLC co-cultures was 7 ± 0.9, n = 4, compared to a CXCR5 MFIR of 91.6 ± 12 for freshly isolated CLL cells the same patients (mean ± SEM, n = 4, p = 0.000). These data indicate that high levels of bioactive CXCL13 are released in NLC cultures that stimulate cognate CXCR5 receptors on CLL B cells and induce signaling cascades, such as p44/42 MAPK, that induce prolonged survival. As such, this study provides a novel insight into interactions between CLL cells and their microenvironment within lymphoid tissues.

scholarly journals HSP90 Stabilizes B-Cell Receptor Kinases in a Multi-Client Interactome: PU-H71 Induces CLL Apoptosis in a Cytoprotective Microenvironment

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5105-5105
Author(s):  
Yue Lynn Wang ◽  
Pin Lu ◽  
Jimmy Lee ◽  
Chao Jie Zhen ◽  
Gabriela Chiosis ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Hsp90 Inhibition ◽  
Apoptotic Pathway ◽  
Induced Apoptosis

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of B-cells in the hematopoietic system and lymphoid tissues. Although inhibitors targeting the B-cell receptor (BCR) pathway have been successful in the treatment of the disease, the underlying mechanisms leading to BCR over-activity in CLL are not fully understood. In this study, we found that HSP90, a highly conserved molecular chaperone, is overexpressed in CLL compared to resting B-cells. HSP90 overexpression is accompanied by the over-expression of several BCR kinases including LYN, SYK, BTK and AKT. Chemical and immune-precipitation demonstrated that these BCR constituents are present in a multi-client chaperone complex with HSP90. Inhibition of HSP90 with PU-H71 destabilized the BCR kinases and caused apoptosis of CLL cells through the mitochondrial apoptotic pathway. Further, PU-H71 induced apoptosis in the presence of stromal co-culture or cytoprotective survival signals. Finally, genetic knock-down of HSP90 client AKT, but not BTK, reduced CLL viability. Overall, our study suggests that the chaperone function of HSP90 contributes to the over-activity of the BCR signaling in CLL and inhibition of HSP90 has the potential to achieve a multi-targeting effect. Thus, HSP90 inhibition may be explored to prevent or overcome drug resistance to single targeting agents. Disclosures No relevant conflicts of interest to declare.

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